In-Depth Proteomics Identifies a Role for Autophagy in Controlling Reactive Oxygen Species Mediated Endothelial Permeability

Endothelial
cells (ECs) form the inner layer of blood vessels and
physically separate the blood from the surrounding tissue. To support
tissues with nutrients and oxygen, the endothelial monolayer is semipermeable.
When EC permeability is altered, blood vessels are not functional,
and this is associated with disease. A comprehensive knowledge of
the mechanisms regulating EC permeability is key in developing strategies
to target this mechanism in pathologies. Here we have used an in vitro
model of human umbilical vein endothelial cells mimicking the formation
of a physiologically permeable vessel and performed time-resolved
in-depth molecular profiling using stable isotope labeling by amino
acids in cell culture mass spectrometry (MS)-proteomics. Autophagy
is induced when ECs are assembled into a physiologically permeable
monolayer. By using siRNA and drug treatment to block autophagy in
combination with functional assays and MS proteomics, we show that
ECs require autophagy flux to maintain intracellular reactive oxygen
species levels, and this is required to maintain the physiological
permeability of the cells.

内皮细胞（Endothelial cells, ECs）构成血管的内层结构，可从物理层面将血液与周围组织分隔开来。为向组织输送营养物质与氧气，内皮单层膜具备半通透性。当内皮细胞通透性发生异常改变时，血管将丧失正常生理功能，这一现象与多种疾病的发生发展密切相关。全面阐明调控内皮细胞通透性的分子机制，是开发针对相关病理过程的靶向干预策略的核心关键。本研究采用模拟生理性通透血管形成的人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVECs）体外模型，并借助细胞培养氨基酸稳定同位素标记（Stable Isotope Labeling by Amino acids in Cell Culture, SILAC）-质谱（mass spectrometry, MS）蛋白质组学技术，开展了时间分辨的深度分子谱分析。研究发现，当内皮细胞组装形成生理性通透的单层结构时，自噬（autophagy）过程会被诱导激活。本研究通过小干扰RNA（small interfering RNA, siRNA）与药物处理联合阻断自噬，并结合功能实验与质谱蛋白质组学分析，证实内皮细胞需要依赖自噬流（autophagy flux）来维持细胞内活性氧（reactive oxygen species, ROS）水平，而这一过程是维持细胞生理性通透性的必要条件。